Development of a quantitative PCR assay to analyze the infection of Tenacibaculum sp. strain Pbs‑1, which is the causative agent of black-spot shell disease

In the year 2018, black-spot shell disease, causing black discoloration of both the shell and pearls, spread widely and significantly impacted the cultured pearl industry. According to our previous study, black-spot shell disease has been attributed to the infection caused by Tenacibaculum sp. Pbs-1. To detect this bacterium specifically, we developed a specific quantitative polymerase chain reaction (qPCR) assay for Tenacibaculum sp. Pbs-1 in the present study. A specific primer set targeted for the internal spacer region (ISR) was designed. Thereafter, the specificity of the primer set was examined with strains of the genus Tenacibaculum. As a result of the optimization of the reaction conditions for qPCR, the original assays we developed were found to have high linearity and quantitative correlations (coefficient of 0.998) for standard plasmid DNA. The assay was able to quantify the strain Pbs-1 when only ten copies of the target ISR were present. Furthermore, we found that the quantification of the strain Pbs-1 was successfully conducted in both the shells of infected pearl oysters and natural seawater from the surrounding environment. These results indicate that our developed quantification method will contribute to clarifying the infection mechanism and assessing the severity of black-spot shell disease.